1. Field of the Invention
The invention relates generally to compositions and methods for detection of a target nucleic acid molecule, and more specifically to compositions and methods for amplifying a signal indicative of a target nucleic acid molecule without the use of enzymes and under isothermal conditions, thus providing for the detection and/or quantitation of a target nucleic acid molecule.
2. Background Information
The polymerase chain reaction (PCR) is the standard molecular biology tool for amplification of DNA molecules. PCR requires a target nucleic acid molecule, a molar excess of forward and reverse primers that bind to the target nucleic acid, deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, and dTTP), and a thermostable DNA polymerase such as Taq polymerase. An amplification reaction is performed under conditions that allow selective hybridization of the forward and reverse primers of an amplification primer pair to the target nucleic acid molecule. Generally, the reaction is performed in a buffered aqueous solution at about pH 7-9.
In a typical PCR amplification cycle, the reaction mixture containing the above is placed in a thermocycler and heated to about 90° C. to 100° C. for about thirty seconds or more. At this temperature the DNA oligonucleotides separate as the hydrogen bonds holding them together break down. The mixture is then cooled down to about 55° C. to about 60° C. At this temperature the forward and reverse primers bind (or anneal) to the single-stranded DNA oligonucleotides. In the final step the mixture is heated again to about 75° C. for at least one minute, which is the optimum temperature for the DNA polymerase enzyme. The polymerase adds bases to the primer segments to build up complementary oligonucleotides of DNA identical to the original molecule. These last three steps can be repeated, for example, up to about thirty times to give about one billion copies of the original DNA. The entire amplification process takes about three hours, with much of the time spent heating and cooling the thermocycler. The amplification products can be detected by standard methods in the art.
There have been many improvements and modification to adapt the original PCR procedure into one that functions as a sensitive detector. However, the nature of most approaches is predominantly biological, as the primary challenges are dependent on the specific properties of the enzymes in question.
Yurke et al., have shown that in the absence of enzymes, DNA are effective fuel-like molecules, and this allows for precise control of movements of DNA in vitro on a nanometer length scale without external prodding (see Yurke et al., “A molecular machine made of and powered by DNA,” pp. 115 (2004), on the world wide web (www) at URL “bell-labs.com/org/physicalsciences/pubs/yurk01.pdf”). Yurke et al. focus on DNA processes such as kinetics of branch migrations, kinetic effects of different binding domain lengths, and breathing rates.
Unfortunately, methods and compositions for detection and amplification of nucleic acids which provide the exponential amplification similar to that of PCR, but takes advantage of the kinetics of nucleic acids to drive the reactions without the use of enzymes and under isothermal conditions, has not been described. Thus, a need exists for methods and materials to detect and amplify nucleic acid molecules in the absence of enzymes, using kinetics of nucleic acids and under isothermal conditions.